1. Field of the Invention
The present invention relates to a plasmidless strain of Bacillus megaterium QM B1551 for use as a bacterial cloning host for recombinant plasmids.
B. megaterium has been the object of study in many laboratories both for its interesting biochemical reactions, and because it germinates synchronously and sporulates more efficiently than most other Bacillus species. Industrially, B. megaterium has been used to produce L-glutamate, pyruvate, cobalamin, single cell protein, and to modify steroids and antibiotics. Up until the present time, however, B. megaterium has not been used as a cloning host to any great extent, a function for which another member of the Bacillus species, Bacillus subtilis, and Escherichia coli have been widely used.
As a cloning host, E. coli has a number of shortcomings which are not common to B. megaterium. For example, E. coli do not secrete proteins in any great quantity and the outer membrane includes an endotoxin. A genetically engineered clone of E. coli must therefore be lysed to recover a protein of interest which is often then contaminated with endotoxin. B. megaterium, on the other hand, is an efficient secretor of protein and has no endotoxins in the cell wall, making it more suitable for mass production of pharmaceuticals. While "safe" strains of E. coli have been developed, "wild" E. coli is a human pathogen whereas B. megaterium like B. subtilis is not.
B. subtilis secretes proteins readily but has two different types of extracellular proteases which digest most foreign protein expressed by the cell, greatly decreasing the yield of the protein of interest. Insofar as known, aall protease negative mutants of B. subtilis are leaky while B. megaterium has only one extracellular protease and some strains are protease negative.
Even though B. megaterium has a number of advantages over E. coIi and B. subtilis, it has a number of disadvantages which have stood in the way of its use as a cloning host. Firstly, it is not as well characterized genetically and, secondly, it contains a large number of naturally-occurring plasmids which potentially might interfere with the expression or further genetic manipulation of any foreign plasmid DNA.
2. Brief Description of the Prior Art
In the past few years, our laboratory has made considerable progress in understanding B. megaterium QM B1551 genetically. We have isolated and characterized a generalized transducing phage for this species, which has been used almost exclusively for mapping in QM B1551, and have mapped in detail several loci. (Vary, P. S., Garbe, J. C., Franzen, M. A. and Frampton, E. W. 1982. MP13, A generalized transducing bacteriophage for Bacillus megaterium. J. Bacteriol. 149:1112-1119; Garbe, J. C. and Vary, P. S. 1981. Bacteriophage MP13 transduction of Bacillus megaterium QM B1551, p. 83 -87. In: Sporulation and Germination. H. S. Levinson, A. L. Sonenshein and D. J. Tripper (eds.), American Society for Microbiology, Washington, D. C.; Callahan, J. P., Crawford, I. P., Hess, G. F. and Vary, P. S. 1983. Cotransductional mapping of the trp-his region of Bacillus megaterium. J. Bacteriol. 154:1112-1116 and Garbe, J. C., Hess, G. F., Franzen, M. A. and Vary, P. S. 1984. 20 Genetics of leucine biosynthesis in Bacillus megaterium. J. Bacteriol. 157:454-459).
In 1980 other laboratories reported protoplast transformation in B. megaterium 216 with a few naturally- occurring plasmids from Bacillus and Staphylococcus. (Brown, B. J. and Carlton, B. C. 1980. Plasmid mediated transformation in Bacillus megaterium. J. Bacteriol. 142:508-512 and Vorobjeva, I. P., Khmel, I. A. and Alfoldi, L. 1980. Transformation of Bacilus megaterium protoplasts by plasmid DNA. FEMS Microbiology Letters. 7:261-263). Thereafter, we began to analyze the resident plasmids in B. megaterium QM B1551, in which all of the genetic mapping had been done with MP13, and to extend the transformation studies to test the stability of foreign plasmids in QM B1551.
In 1984, we published an article (Kieselburg, M. K., Weikert, M. and Vary, P. S. Analysis of resident and transformant plasmids in Bacillus megaterium. Bio/Technology. 2:254-259) reporting that the resident plasmids of QM B1551 had been analyzed and that several Bacillus cloning plasmids had been successfully transformed into QM B1551 by protoplast fusion. More particularly, we analyzed the plasmid array of B. megaterium QM B1551 by sucrose gradient centrifugation, agarose gel electrophoresis and electron microscopy measurements and found seven plasmid sizes ranging in molecular weight from 3.5 to 109.times.10.sup.6. The plasmids transformed by protoplast fusion were found to be stable and present in high copy number suggesting that B. megaterium QM B1551 might be a desirable cloning host.
Since the use of transposons greatly increases the genetic versatility of an organism, we continued our work with B. megaterium QM B1551 by testing whether a transposon could be introduced into QM B1551. In 1986, we reported that transposon Tn917, carried on plasmid pTV1, had been successfully introduced into QM B1551 and transposed efficiently and apparently without hot spots. (Bohall, N. A. and Vary, P. S. Transposition of Tn917 in Bacillus megaterium. J. Bacteriol. 167:716-718).
Because at least 11% of the cellular DNA of B. megaterium QM B1551 is present as plasmid DNA, it seemed unlikely that QM B1551 had much prospect for use industrially as a cloning host unless it could be cured of its plasmids. Having proved the stability of foreign plasmids in B. megaterium QM B1551 and the use of transposons, our attention was now directed to the development of a plasmidless strain.
In earlier work, another laboratory isolated a plasmidless strain of B. megaterium 19213, designated VT1600 (ATCC 35985) (Von Tersch, M. A. and Carlton, B. C. 1984. Molecular cloning of structural and immunity genes for megacins A-216 and A-19213 in B. megaterium. J. Bacteriol. 160:854-859) but they did not test for expression of recombinant proteins and B. megaterium 19213 is much less well characterized genetically than QM B1551.
Our initial attempts to cure B. megaterium QM B1551 of its plasmids were only partly successful. In a thesis accepted on June 4, 1985, we reported that sublethal concentrations of novobiocin and ethidium bromide produced a number of strains, some of which were cured of all but a few plasmids, but all of which contained some plasmids. (Katherine Lee Weiland, M. S. Thesis. Department of Biological Sciences. Northern Illinois University. Plasmid analysis of megacin negative strains of Bacillus megaterium QM B1551). Hence, at the time the present invention was made we were doubtful whether a plasmidless strain of QM B1551 could be obtained and, if obtained, whether it would be genetically altered in other ways in view of the large amount of DNA being removed from the cell.
In view of the above, it is an object of the present invention to provide a plasmidles strain of B. megaterium QM B1551. It is another object to provide a plasmidless strain of B. megaterium QM B1551 in which, like the parent stock, foreign plasmids are stable and transposons can be introduced efficiently and randomly. It is still another object of the present invention to provide a plasmidless strain which retains the parent stock's neutral protease. Other objects and features of the invention will be in part apparent and in part pointed out hereinafter, the scope of the invention being indicated by the subjoined claims.